Introduction

With the ever-increasing acceptance of combination therapy and the prevalence of chronic diseases in the world, the use of traditional Chinese medicine (TCM) has become an emerging trend1. A TCM prescription usually contains numerous ingredients synergistically and holistically acting on the diseases. Herb pairs, the simplest form and the centralized representative of Chinese herbal compatibility, intrinsically convey the basic idea of TCM prescriptions2. Danggui and Honghua combined as an herb pair have been frequently used in TCM prescriptions, which could be ascended to an ancient and classical formula Danggui-Honghua Decoction. Danggui, the radix of Angelica sinensis (Oliv.) Diels, has been used for thousands of years in the East Asia and was firstly recorded in a classical masterpiece of TCM Shennong Bencao **g (200–300 A.D., Han Dynasty). Danggui has also been called “female ginseng”, predominantly renowned for treatment of intractable gynecological disorders31, and databases were mined including Herbal Ingredients’ Targets database (HIT, http://lifecenter.sgst.cn/hit/)32, Therapeutic Targets Database (TTD, http://bidd.nus.edu.sg/group/ttd/)33, DrugBank (http://www.drugbank.ca/) and Google Scholar. Finally, 42 targets related to BSS were determined, interacting with the selected 31 active ingredients of this herb pair (Table 2).

Table 2 Target information of Danggui-Honghua.
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Target proteins of Danggui

Thirty-three targets were identified for 15 active ingredients of Danggui with 108 interactions. Multiple therapeutic targets concerning BSS were mediated by the active ingredients of Danggui, such as ESR1, ESR2, PTGS1, PTGS2, JUN, ICAM1, NOS2, NOS3, and MAPK1. Most of these targets are involved in vascular and central neural systems. For instances, ESR1 and ESR2 mediate the vascular system to promote the functional recovery of vascular injury and provide neuroprotective effects in central neural system34; PTGS1 and PTGS2 contribute to atherosclerosis and thrombosis by regulating the production of eicosanoids that modulate physiological processes in the vessel wall35; JUN modulates smooth muscle cell proliferation in response to vascular angioplasty36; additionally, ICAM1 mediates the adhesion of neutrophils and monocytes to vascular endothelium37. Specifically, Z-ligustilide, senkyunolide I, ferulic acid and coniferyl ferulate may mediate NOS2 or NOS3 to increase the nitric oxide biosynthesis, thereby possibly exerting blood-vessel dilation, neuronal signal transmission, coordination of heart rhythm and regulation of cellular respiration activities38. Vanillin may interact with four potential targets including PTGS2, JUN, MMP9 and MAPK1 which are also relevant to nervous system and vascular diseases39.

Except for vanillin, nicotinic acid and β-sitosterol, other active ingredients from Danggui also interacted with the targets related to inflammation, abdominal pain, dysmenorrhea, embolism, thrombosis and ischemia. Six potential targets including F2, F7, F10, F11, TBXA2R and MAPK14 are relevant to disorders of thrombosis, which could clarify why Danggui exhibits strong anticoagulant function16. Additionally, there are also several targets such as PPARG, SOD1, RELA and GSK3B are involved in the ischemic and inflammation processes. It should be pointed out that some major targets, such as PTGS2, NOS3 and CHRM2, are also closely concerned with the pain-related diseases, which may contribute to the blood-activating and stasis-dissolving effects of Danggui.

Target proteins of Honghua

For Honghua, by target fishing, 16 active ingredients were validated to bind with 36 target proteins related to BSS. There are also plenty targets mediated by the active ingredients from Honghua that are involved in vascular and central neural systems. For example, HSYA may have the potential to act on 18 targets including HIF1A, VEGFA, HMOX1, PTGS2, CASP3, CASP9, AGTR1, PTAFR, and GSK3B. Actually, HSYA has been identified as an inhibitor of HIF1A, which might contribute to its therapeutic application in vascular diseases40. Beyond that, it also has a strong antagonistic effect on the PTAFR protein, which may explain its function in both inflammatory and neuropathic pain responses41. Analogously, scutellarin exhibits strong neuroprotective effect because it may interact with STAT1, NOS2, NOS3, VEGFA and FGFR1 simultaneously42.

Inflammatory response is of enormous significance in BSS. Six active compounds of Honghua, including 6-hydroxykaempferol (HH-6), eriodictyol (HH-11), rosmarinic acid (HH-16), kaempferol, quercetin, and luteolin, may have interactions with PPARG which is expected to control inflammation associated with gut, myocardial, lung and cerebral ischemia43. Other five active ingredients, HSYA, rutin (HH-10), safflor yellow A (HH-2), safflor yellow B and eriodictyol, were identified to interact with PTGS1, which is a potential target for the next generation of anti-inflammatory drugs44.

Of note, we have implemented molecular docking combined with the in vitro experiments to explore the structure-activity relationships of the active ingredients among Si-Wu-Tang series containing the herb pair Danggui-Honghua45,46. Flavonoids including quercetin (IC50 = 0.035 mM), luteolin (IC50 = 0.052 mM), kaempferol (IC50 = 0.109 mM), and acacetin (IC50 = 0.140 mM) showed F2 inhibition activity in vitro. By the software of Molegro Virtual Docker (http://www.molegro.com), quercetin (−89.72 kJ/mol) exhibited high average MolDock score value and interacted with the active site residues Ala230, His79, Lys88, Gly258, Ser256, and Trp86 of F245. It was also found that ferulic acid (60.45%), quercetin (70.00%), kaempferol (45.15%), HSYA (45.28%), rutin (90.83%), and scutellarin (53.46%) in 20 μg/mL showed higher estrogenic activity in vitro than caffeic acid (24.73%) in 20 μg/mL. And ferulic acid (−72.8 kJ/mol), quercetin (−76.3 kJ/mol) and rutin (−87.2 kJ/mol) exhibited high average MolDock score values and strong binding affinity to ESR146. It could be concluded that the predicted targets were in agreement with our previous experiments. Therefore, the integrated in silico approach should be feasible and convincible to explore the compound-target interactions of Danggui-Honghua.

Target and contribution index analysis to decipher the combination rule of Danggui-Honghua

To facilitate the visualization and interpretation of the complex relationships between all active ingredients of Danggui-Honghua and their targets, a bipartite graph of C-T network was constructed (Fig. 3). Two characteristics of Danggui-Honghua were observed from network analysis: (1) the promiscuous properties of its active ingredients and (2) the existence of highly interconnected compounds. The average number of potential targets per active ingredient was 6.5. And all active ingredients in this herb pair were potential multiple-kinase inhibitors or activators. Amongst them, those ones with high interconnection degrees were responsible for the high interconnectedness of the C-T network, especially HSYA (degree = 18), Z-ligustilide (degree = 15), quercetin (degree = 12), luteolin (degree = 11), ferulic acid (degree = 11) and Z-butylidenephthalide (degree = 10). From the topological features of this network and the functional properties of the proteins (Supplementary Table S2 and Fig. S1A), there were different modes of actions between compounds and targets in Danggui-Honghua. As shown in the C-T network (Fig. 3), the efficacy of this herb pair not only concentrated on modulating the crucial targets involving in the vascular and central neural systems (ESR1, ICAM1, HMOX1 and NOS3), but also, more essentially, focused on the regulation of the other proteins mediating inflammation, thrombosis, ischemia, dysmenorrhea and abdominal pain (F7, TBXA2R, PTGS2, CHRM2, and NOS2) to relieve the pathological changes and prolong the efficient curing process. For example, in animal models, increased expression of HMOX1 has been shown to protect tissues and cells against ischemia-reperfusion injury, oxidative stress, inflammation and hypoxia-induced vascular stasis47. The protein HMOX1 was found to have interactions with 9 ingredients, and senkyunolide A, kaempferol and eriodictyol synergistically increase HMOX1 expression48,49,50. In addition, the neurotoxic proinflammatory mediator PTGS2 was also connected with several active ingredients. Especially, HSYA, Z-ligustilide and ferulic acid were confirmed to enhance anti-inflammatory effects via significantly attenuatingthe expression level of PTGS24,51,52.

Figure 3: Compound-target network of Danggui-Honghua.
figure 3

Thirty-one active compounds (HH-/DG-, rectangle) map 42 potential protein targets (short name referred in Table 2, circle). The 15 bright green rectangles are active compounds from Danggui and the 16 red ones represent those from Honghua. The 27 orange circles are the proteins targeted by those compounds screened out of both herbs. The 6 dark pink circles on the upper side are the potential proteins hit by the compounds of Danggui and 9 tea green ones on the bottom side are the potential proteins only targeted by the compounds of Honghua.

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The T-D network was further constructed based on all the targets and their corresponding diseases. As shown in the graphical network (Fig. 4), both Danggui and Honghua could regulate the proteins related to vascular and central neural system diseases as well as inflammation and pain. The target scope of Honghua was broader than that of Danggui. Several targets mediated by the active ingredients of Honghua including PTAFR, PTGS2, NOS3 and STAT1, may alleviate the accompanying symptoms of BSS such as inflammation and pain. As to Danggui, its active ingredients mediated several targets including ADRB1, CHRM2, GSK3B, BCL2 and ICAM1, which may be helpful for improving the immune system. Modern research has demonstrated that ischemic processes release mediators activating the innate immune system that may be induced by the formation of thrombi inside blood vessels53. Fortuitously, several targets associated with cancer were also discerned in the T-D network. HSYA from Honghua was validated to antagonize tumor angiogenesis by inhibiting the protein expression of VEGFA, MMP9 and HIF1A4. Additionally, Z-butylidenephthalide, senkyunolide A and Z-ligustilide from Danggui were confirmed to exhibit anti-proliferative potential and significant synergy on colon cancer cells54.

Figure 4: Target-disease network of Danggui-Honghua.
figure 4

The 6 purple circles and 9 red circles represent the targets of Danggui and Honghua, respectively. The 27 pink circles represent the targets shared by Danggui and Honghua. The 7 yellow diamonds represent the disorders related to those 42 targets.

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As mentioned earlier, a contribution index of every active ingredient was proposed based on network based efficacy weighted by literature. According to calculated results (Fig. 5 and Supplementary Table S3), six compounds emerged from the active ingredients, including HSYA, safflor yellow A, safflor yellow B, Z-ligustilide, ferulic acid, and Z-butylidenephthalide. They displayed the most contribution to the blood-activating and stasis-dissolving effects of Danggui-Honghua with a sum of CIs of 88.13%. Therefore, the above discussion may fully clarify why Danggui-Honghua could produce synergistic and complementary effects.

Figure 5
figure 5

The CI and accumulative CI of active ingredients in Danggui-Honghua.

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Pathway analysis to explore the underlying mechanisms of Danggui-Honghua

Signaling pathways, as an important component of the system pharmacology, link receptor-ligand interactions to pharmacodynamics outputs55. The canonical pathways associated with BSS treatment and prophylaxis were extracted from Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) database, which ends up with 20 KEGG pathways, including cAMP signaling pathway, calcium signaling pathway, NF-κB signaling pathway and sphingolipid signaling pathway (Fig. 6). The NF-κB signaling pathway is crucial for focal cerebral ischemia/reperfusion induced inflammatory injury56. Previous research has shown that HSYA, safflor yellow A, and caffeic acid could inhibit the NF-κB signaling pathway contributing to the cross-talk of multiple targets in anti-inflammation57. The sphingolipid signaling pathway was proven to play a critical role in the ischemic preconditioning and the pathophysiology of stroke58.

Figure 6: Distribution of partial targets of Danggui-Honghua on the compressed pathway.
figure 6

The orange nodes are potential targets. And the light blue nodes are relevant targets in the pathway.

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For the purpose of systematically dissecting the underlying mechanisms of Danggui-Honghua, all of the targets interacting with the active ingredients were mapped onto the 20 KEGG pathways and the T-P network was generated (Fig. 7). The TNF signaling pathway exhibited the highest number of target connections (degree = 11), followed by HIF-1 signaling pathway with 9 targets, estrogen signaling pathway and neurotrophin signaling pathway with 8 ones, respectively. These high-degree pathways were closely related to the vascular and central neural systems and inflammation. The TNF signaling pathway plays an important role in the ischemic stroke and the vascular injury involved in multiple targets including JUN, PTGS2 and ICAM159,60. The HIF-1 signaling pathway was an underlying mechanism of neuroprotection and anti-ventricular cell apoptosis61,62. Fortunately, 22 out of 31 active compounds from Danggui-Honghua, especially HSYA, quercetin, Z-ligustilide and luteolin, were implicated in regulating the major targets of HIF-1 signaling pathway, such as HMOX1, NOS2, NOS3, RELA, PIK3CG and MAPK1. Recently, we successfully discovered the underlying blood-activating mechanisms of Danggui-Honghua by metabolomics analysis. Four potential metabolic pathways were speculated, including phenylalanine metabolism, sphingolipid metabolism, arachidonic acid metabolism, and arginine and proline metabolism63. In the present study, arginine and proline metabolism, arachidonic acid metabolism, and sphingolipid signaling pathway (including sphingolipid metabolism) were also deciphered, which were consistent with our previous metabolomics results63.

Figure 7
figure 7

Target-pathway network of Danggui-Honghua where light green nodes represent the targets and purple nodes the pathways.

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Overall, it could be speculated that the herb pair Danggui-Honghua exert the blood-activating and stasis-dissolving effects mainly through the regulation of TNF, HIF-1, estrogen, and neurotrophin signaling pathways. As a holistic medicine, this herb pair may also be implicated in arginine and proline metabolism, arachidonic acid metabolism, VEGF signaling pathway, MAPK signaling pathway, calcium signaling pathway, and sphingolipid signaling pathway to regulate the vascular and nervous systems, as well as the inflammation and pain.

Discussion

Herb pairs, the smallest compatible units in TCM formulae, have become a prominent concern during the past decade2. Danggui and Honghua, combined as a classical herb pair, have been frequently used in TCM prescriptions5. Through data mining and pharmacological approaches, this herb pair was found to produce synergistic and complementary effects to treat BSS64,47,48,49,50,51,73. Subsequently, 20 signaling pathways associated with BSS treatment and prophylaxis by Danggui-Honghua were retrieved. A recent study on the anti-inflammatory effects of volatile oils from Danggui (78.61% Z-Ligustilide and 7.99% Z-Butylidenephthalide) using GC-MS-based metabolomics revealed that it may be involved in regulating the arachidonic acid metabolism74, which is comparable to our findings. Finally, the C-T, T-D and T-P networks clearly elucidated the molecular synergistic actions of Danggui-Honghua in a holistic context. By network systematic analysis and contribution index calculation, HSYA, safflor yellow A and safflor yellow B in Honghua, together with Z-ligustilide, ferulic acid and Z-butylidenephthalide in Danggui, displayed the most contribution to the blood-activating and stasis-dissolving effects of Danggui-Honghua. Meanwhile, this herb pair could regulate the proteins related to vascular and central neural system diseases as well as inflammation and pain implicated in TNF, HIF-1, estrogen, and neurotrophin signaling pathways, arginine and proline metabolism, arachidonic acid metabolism, and VEGF signaling pathway. Noteworthy, based on our research, several targets and signaling pathways of Danggui-Honghua acting on BSS have been found for the first time. All of these results are expected to help identify novel curative efficacy and take full clinical advantage of Danggui-Honghua.

Due to the above findings mainly relied on theoretical analyses, more experiments are anticipated to support these findings as well as potential clinical applications. It should be noted that the majority of the ingredients in both Danggui and Honghua were hydrotropic, and the OB values of a third of the selected 31 active ingredients were less than 30%. Therefore, the availability of these active constituents by gut microbiota especially under the disease state may be a critical step towards the emergence of their bioactivities in vivo75.

Methods

Chemical ingredients database building

All of the constituent data of Danggui (the radix of A. sinensis) and Honghua (the florets of C. tinctorius) were retrieved from TcmSP™, a unique system pharmacology platform designed for herbal medicines12, and then manually supplemented through a wide-scale text-mining method. Meanwhile, four important pharmacology-related properties were also obtained from TcmSP™, including MW, MLogP, nHDon and nHAcc.

Active ingredients screening

The active ingredients from Danggui-Honghua were filtered by integrating three indexes including OB, Caco-2, and DL. A robust in silico model OBioavail 1.1 (Yangling, Shaanxi, China) that integrated the metabolism and transport information was employed to calculate the OB values of all herbal ingredients76. Those ingredients with OB ≥ 30% were selected. The VolSurf built-in Caco-2 permeability model (Tripos, St. Louis, America)77 was implemented to screen active compounds. Given that molecule with Caco-2 value less than −0.4 is not permeable, the threshold of Caco-2 permeability was set to −0.4. Database-dependent DL evaluation approach based on Tanimoto coefficient31. Amongst them, the PharmMapper gives the best map** poses by comparing with available targets in PharmTargetDB (a large, in-house repertoire of pharmacophore database belongs to PharmMapper) and the respective N-best fit poses are generated. All active compounds were also sent to HIT32, TTD33, DrugBank and Google Scholar to mine compound-target interactions supported by literature. Then, to better dissect the role of Danggui-Honghua in BSS treatment, all targets obtained from the previous two steps were sent to TTD, Comparative Toxicogenomics Database (CTD, http://ctdbase.org/) and PharmGKB (http://www.pharmgkb.org)79 to mine target-related diseases. Finally, those targets which were implicated in the aforementioned pathophysiology and clinical manifestations of BSS were retained, and the others were eliminated.

Networks construction

Three networks were constructed: (1) Compound-target network (C-T network). Active ingredients of Danggui-Honghua and their corresponding targets were employed to generate the C-T network. (2) Target-disease network (T-D network). All targets and their corresponding diseases were employed to build a bipartite graph of T-D network. (3) Target-pathway network (T-P network). The pathway information of targets were extracted from the database of KEGG, and then a bipartite T-P network composed of targets and their corresponding putative pathways was built. All visualized network graphs were constructed by Cytoscape 3.2.1 (http://www.cytoscape.org/), an open software package project for visualizing, integrating, modeling and analyzing the interaction networks80.

Contribution indexes calculation

In order to estimate the contribution of each active ingredient to the blood-activating and stasis-dissolving effects of Danggui-Honghua, a contribution index (CI) based on network based efficacy (NE) weighted by literature was proposed and calculated by eqs (2) and (3):

where n is the number of targets associated with ingredient j; di is the degree of target i associated with ingredient j; ci is the number of BSS-related literature of ingredient i; m is the number of ingredients.

If the sum of CIs for the top N ingredients was more than 85%, these relevant N ingredients were considered to contribute the most to the blood-activating and stasis-dissolving effects.

Additional Information

How to cite this article: Yue, S.-J. et al. Herb pair Danggui-Honghua: mechanisms underlying blood stasis syndrome by system pharmacology approach. Sci. Rep. 7, 40318; doi: 10.1038/srep40318 (2017).

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